This invention relates to thrombin inhibitors which are useful anticoagulants in mammals. In particular it relates to heterocyclic derivatives having high anticoagulant activity, and antithrombotic activity. Thus, this invention relates to new inhibitors of thrombin, pharmaceutical compositions containing the compounds as active ingredients, and the use of the compounds as anticoagulants for prophylaxis and treatment of thromboembolic disorders such as venous thrombosis, pulmonary embolism, arterial thrombosis, in particular myocardial ischemia, myocardial infarction and cerebral thrombosis, general hypercoagulable states and local hypercoagulable states, such as following angioplasty and coronary bypass operations, and generalized tissue injury as it relates to the inflammatory process. In addition, the antithrombotic agents are useful as anticoagulants in in vitro applications.
The process of blood coagulation, thrombosis, is triggered by a complex proteolytic cascade leading to the formation of thrombin. Thrombin proteolytically removes activation peptides from the Axcex1-chains and the Bxcex2-chains of fibrinogen, which is soluble in blood plasma, initiating insoluble fibrin formation.
Anticoagulation currently is achieved by the administration of heparins and coumarins. Parenteral pharmacological control of coagulation and thrombosis is based on inhibition of thrombin through the use of heparins. Heparins act indirectly on thrombin by accelerating the inhibitory effect of endogenous antithrombin III (the main physiological inhibitor of thrombin). Because antithrombin III levels vary in plasma and because clot-bound thrombin seems resistant to this indirect mechanism, heparins can be an ineffective treatment. Because coagulation assays are believed to be associated with efficacy and with safety, heparin levels must be monitored with coagulation assays (particularly the activated partial thromboplastin time (APTT) assay). Coumarins impede the generation of thrombin by blocking the posttranslational gamma-carboxylation in the synthesis of prothrombin and other proteins of this type. Because of their mechanism of action, the effect of coumarins can only develop slowly, 6-24 hours after administration. Further, they are not selective anticoagulants. Coumarins also require monitoring with coagulation assays (particularly the prothrombin time (PT) assay).
Recently, interest has grown in small synthetic molecules which demonstrate potent direct inhibition of thrombin. See, for example Robert M. Scarborough, Annual Reports in Medicinal Chemistry, (1995), 30, 71-80.
Although the heparins and coumarins are effective anticoagulants, no commercial drug has yet emerged from the small synthetic molecules; and despite the continuing promise for this class of compounds, there still exists a need for anticoagulants which act selectively on thrombin, and which, independent of antithrombin III, exert inhibitory action shortly after administration, preferably by an oral route, and do not interfere with lysis of blood clots, as required to maintain hemostasis.
The present invention is directed to the discovery that the compounds of the present invention, as defined below, are potent thrombin inhibitors that may have high bioavailability and favorable pharmacokinetics following oral administration.
According to the invention there is provided a method of inhibiting thrombin comprising using an effective amount of a thrombin inhibiting compound of formula I (or a pharmaceutically acceptable salt thereof) 
wherein
E is CH or CRe in which Re is methyl, methoxy or halo;
R denotes 0, 1 or 2 substituents on the benz-ring independently selected from halo, methyl, ethyl, hydroxy, methoxy, carbamoyl, aminomethyl and hydroxymethyl;
R1 is R1a, R1b, or R1c in which
R1a is xe2x80x94CH2xe2x80x94Rr, in which Rr is 5-tetrazolyl, 2-carboxypyrrolidin-1-yl or 2-[[(1-4C)alkoxy]carbonyl]-pyrrolidin-1-yl; 2-carboxy-5-oxopyrrolidin-1-yl or 2-[[(1-4C)alkoxy]carbonyl]-5-oxopyrrolidin-1-yl;
R1b is xe2x80x94X1xe2x80x94(CH2)sxe2x80x94NRsRt in which X1 is a direct bond, methylene or O; s is 1 or 2; provided that when s is 1, then X1 is a direct bond, and further provided that the chain xe2x80x94(CH2)sxe2x80x94 may bear one or two methyl or ethyl substituents or may be part of a trans-1,2-cyclohexanediyl; and Rs and Rt are independently hydrogen or (1-3C)alkyl or the group NRsRt is pyrrolidino, piperidino, morpholino, 1-imidazolyl, 1-pyrazolyl, 1,2,4-triazol-1-yl, or benzylamino; and
R1c is xe2x80x94X1xe2x80x94(CH2)sxe2x80x94NRsRt in which X1 is a direct bond, methylene or O; s is 1 or 2; provided that when s is 1, then X1 is a direct bond, and further provided that the chain xe2x80x94(CH2)sxe2x80x94 may bear one or two methyl or ethyl substituents or may be part of a trans-1,2-cyclohexanediyl; and the group NRsRt is 2-oxopyrrolidin-1-yl, 2,5-dioxopyrrolidin-1-yl, 2-oxooxazolidin-3-yl, 2-oxoimidazolidin-1-yl, 3-methyl-2-oxoimidazolidin-1-yl, 2-oxopyrrolidin-3-yl, 1-methyl-2-oxopyrrolidin-3-yl, 1-tetrazolyl, methylsulfonylamino or phenylsulfonylamino; and
R2 is R2a, R2b, or R2c in which
R2a is xe2x80x94X2xe2x80x94(CH2)nxe2x80x94Rf in which X2 is a direct bond, methylene or O; n is 1, 2 or 3; and Rf is 5-tetrazolyl, carboxy, [(1-4C)alkoxy]carbonyl or hydroxymethyl; or (provided that when n is 1, X2 is a direct bond) Rf is 2-carboxypyrrolidin-1-yl, 2-[[(1-4C)alkoxy]carbonyl]pyrrolidin-1-yl, (carboxymethyl)amino, [[(1-4C)alkoxy]carbonylmethyl]amino, (4-carboxymethylimidazol-1-yl)amino, [4-[[(1-4C)alkoxy]carbonylmethyl]imidazol-1-yl]amino, (4-carboxybenzyl)amino, [4-[[(1-4C)alkoxy]carbonyl]benzyl]amino, (3-amino-1,4-dioxo-4-hydroxybutyl)amino or [3-amino-1,4-dioxo-[(1-4C)alkoxy]butyl]amino;
R2b is xe2x80x94X2xe2x80x94(CH2)mxe2x80x94NRaRb in which X2 is a direct bond, methylene, O or S; m is 1, 2, 3, 4 or 5; provided that when m is 1, then X2 is a direct bond; and Ra and Rb are independently hydrogen, or (1-3C)alkyl, or one of Ra and Rb is hydrogen or methyl and the other is t-butyl, benzyl, or pyridylmethyl; or the group NRaRb is pyrrolidino, piperidino, morpholino, 1-imidazolyl, 1-pyrazolyl, or 1,2,4-triazol-4-yl; or
R2b is xe2x80x94[X2xe2x80x94(CH2)n]pxe2x80x94N(Ra)xe2x80x94COxe2x80x94A in which X2 is a direct bond, methylene or O; n is 1, 2, 3 or 4; p is 0 or 1, Ra is hydrogen or methyl; and xe2x80x94COxe2x80x94A is a natural or unnatural xcex1-amino acyl group, which may bear one or more pharmaceutically acceptable protecting groups and may be further substituted on the xcex1-nitrogen; and
R2c is hydrogen, or
R2c is xe2x80x94NRaxe2x80x94COxe2x80x94(CH2)mxe2x80x94Rb or xe2x80x94Oxe2x80x94CH2xe2x80x94Rb in which m is 0 or 1, Ra is hydrogen or methyl, and Rb is a ring of formula XII or formula XIII 
xe2x80x83in which G is O, S, NH or CH2 and Rc is hydrogen or methyl, and L is NRf or CH2 and Rf is hydrogen or methyl; or
R2c is xe2x80x94NHCORg in which Rg is a five-membered heteroaromatic ring having 2 heteroatoms selected from O, S and N and in which the carbonyl group is bonded to a ring carbon situated between a ring heteroatom and another ring carbon; or
R2c is xe2x80x94(CH2)nxe2x80x94Rh, xe2x80x94Oxe2x80x94(CH2)nxe2x80x94Rh or xe2x80x94NHxe2x80x94(CH2)nxe2x80x94Rh in which n is 0, 1 or 2 and Rh is cyclopentyl, cyano, or xe2x80x94CONRiRj in which Ri and Rj are independently hydrogen or methyl or the group NRiRj is pyrrolidino, piperidino, or morpholino; or
R2c is xe2x80x94X2xe2x80x94(CH2)pxe2x80x94Rk, or xe2x80x94Oxe2x80x94CH2xe2x80x94CH(CH3)xe2x80x94Rk in which X2 is a direct bond, methylene or O and p is 1, 2 or 3, provided that when p is 1, then X2 is a direct bond, and Rk is 2-oxopyrrolidin-1-yl or NHCORm in which Rm is (1-3C)alkyl, phenyl or pyridyl; or
R2c is xe2x80x94NHxe2x80x94COxe2x80x94NRiRj in which Ri and Rj are independently hydrogen or methyl or the group NRiRj is pyrrolidino, piperidino, or morpholino; or
R2c is xe2x80x94Oxe2x80x94COxe2x80x94NRpRq in which Rp and Rq are independently hydrogen, methyl or ethyl or the group NRpRq is pyrrolidino, piperidino, or morpholino; or
R2c is xe2x80x94NHxe2x80x94SO2xe2x80x94Rr in which Rr is (1-3C)alkyl or phenyl; and
provided that either R1 is R1b or R2 is R2b.
The xcex1-amino acyl group xe2x80x94COxe2x80x94A conveniently may be represented as xe2x80x94COxe2x80x94CH(Rb)xe2x80x94NRfRg, or may be denoted by standard amino acid nomenclature. Thus, xe2x80x94COxe2x80x94A may be an xcex1-amino acyl group derived from an xcex1-amino acid selected from glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, serine, threonine, methionine, cysteine, proline, azetidine-2-carboxylic acid, pipecolic acid, aspartic acid, asparginine, glutamic acid, glutamine, lysine, arginine, histidine, etc. in which an amino group may bear, for example, a t-butoxycarbonyl protecting group; a carboxy group may be protected as its (1-4C)alkyl ester; a hydroxy group may bear, for example, a benzyl protecting group; and a thiol group may bear, for example, a t-butyl protecting group. In addition, when xe2x80x94COxe2x80x94A is represented as xe2x80x94COxe2x80x94CH(Rb)xe2x80x94NRfRg, each of Rf and Rg may be hydrogen or methyl, or xe2x80x94NRfRg may be a pyrrolidino, piperidino, morpholino or 1,1-dioxothiomorpholin-4-yl group (and Rb denotes the side chain or protected side chain of an xcex1-amino acyl group as defined above).
A particular compound of formula I is a compound of formula Ia 
wherein
E is CH or CRe in which Re is methyl, methoxy or halo;
R1 is xe2x80x94X1xe2x80x94(CH2)sxe2x80x94NRsRt in which X1 is a direct bond, methylene or O; s is 1 or 2; provided that when s is 1, then X1 is a direct bond; and Rs and Rt are independently hydrogen or (1-3C)alkyl or the group NRsRt is pyrrolidino, piperidino, morpholino, 1-imidazolyl, 1-pyrazolyl, 1,2,4-triazol-1-yl, or benzylamino;
R2 is hydrogen or xe2x80x94X2xe2x80x94(CH2)mxe2x80x94NRaRb in which X2 is a direct bond, methylene, O or S; m is 1, 2, 3, 4 or 5; provided that when m is 1, then X2 is a direct bond; and Ra and Rb are independently hydrogen or (1-3C)alkyl or the group NRaRb is pyrrolidino, piperidino, morpholino, 1-imidazolyl, 1-pyrazolyl, 1,2,4-triazol-4-yl, or 2-oxopyrrolidin-1-yl; and
R5 is hydrogen, hydroxy or methoxy.
A particular value for xe2x80x94X1xe2x80x94(CH2)sxe2x80x94NRsRt is xe2x80x94CH2xe2x80x94NRsRt in which NRsRt is pyrrolidino, 1-imidazolyl, 1-pyrazolyl, 1,2,4-triazol-1-yl, or benzylamino or is xe2x80x94Oxe2x80x94(CH2)2xe2x80x94NRsRt in which NRsRt is pyrrolidino, and more particularly, is xe2x80x94CH2xe2x80x94NRsRt.
A particular value for R2 is xe2x80x94Oxe2x80x94(CH2)2xe2x80x94NRaRb in which NRaRb is pyrrolidino, 1-pyrazolyl or 2-oxopyrrolidin-1-yl. A particular value for R5 is hydroxy.
The present invention also provides a method of inhibiting coagulation in a mammal comprising administering to a mammal in need of treatment, a coagulation inhibiting dose of a thrombin inhibiting compound of formula I having any of the above definitions.
The present invention further provides a method of inhibiting thrombin comprising administering to a mammal in need of treatment, a thrombin inhibiting dose of a thrombin inhibiting compound of formula I having any of the above definitions.
Further, the present invention provides a method of treating a thromboembolic disorder comprising administering to a mammal in need of treatment, an effective dose of a thrombin inhibiting compound of formula I having any of the above definitions.
In addition, there is provided the use of a thrombin inhibiting compound of formula I having any of the above definitions for the manufacture of a medicament for treatment of a thromboembolic disorder.
As a further aspect of the invention, there is provided a prodrug (or a pharmaceutically acceptable salt thereof) of any of the above described thrombin inhibiting compounds of formula I which will form a prodrug. (It will be recognized that a thrombin inhibiting compound of formula I also may serve as a prodrug for a different thrombin inhibiting compound of formula I).
As an additional feature of the invention there is provided a pharmaceutical formulation comprising in association with a pharmaceutically acceptable carrier, diluent or excipient, a prodrug of a thrombin inhibiting compound of formula I (or of a pharmaceutically acceptable salt thereof) as provided in any of the above descriptions.
In general, the thrombin inhibiting compounds of formula I are believed to be novel and, thus, to constitute an additional aspect of the invention. Thus, according to the invention there is provided a novel compound of formula I (or a pharmaceutically acceptable salt thereof) according to any of the above definitions of a compound of formula I, provided that the compound is not one which is not novel A pharmaceutically acceptable salt of an antithrombotic agent of the instant invention includes one which is an acid-addition salt made with an acid which provides a pharmaceutically acceptable anion or one which is a salt made with a base which provides a pharmaceutically acceptable anion. Examples of such acids are provided hereinbelow. Thus, a pharmaceutically acceptable salt of a novel compound of formula I as defined above provides a particular aspect of the invention.
As an additional aspect of the invention there is provided a pharmaceutical formulation comprising in association with a pharmaceutically acceptable carrier, diluent or excipient, a novel compound of formula I (or a pharmaceutically acceptable salt thereof) as provided in any of the above descriptions.
In this specification, the following definitions are used, unless otherwise described: Halo is fluoro, chloro, bromo or iodo. Alkyl, alkoxy, etc. denote both straight and branched groups; but reference to an individual radical such as xe2x80x9cpropylxe2x80x9d embraces only the straight chain (xe2x80x9cnormalxe2x80x9d) radical, a branched chain isomer such as xe2x80x9cisopropylxe2x80x9d being specifically denoted.
It will be appreciated that certain compounds of formula I (or salts or prodrugs, etc.) may exist in, and be isolated in, isomeric forms, including cis- or trans-isomers, as well as optically active, racemic, or diastereomeric forms. It is to be understood that the present invention encompasses a compound of formula I as a mixture of diastereomers, as well as in the form of an individual diastereomer, and that the present invention encompasses a compound of formula I as a mixture of enantiomers, as well as in the form of an individual enantiomer, any of which mixtures or form possesses inhibitory properties against thrombin, it being well known in the art how to prepare or isolate particular forms and how to determine inhibitory properties against thrombin by standard tests including those described below.
In addition, a compound of formula I (or salt or prodrug, etc.) may exhibit polymorphism or may form a solvate with water or an organic solvent. The present invention also encompasses any such polymorphic form, any solvate or any mixture thereof.
Particular values are listed below for radicals, substituents, and ranges, for illustration only, and they do not exclude other defined values or other values within defined ranges for the radicals and substituents.
A particular value for a (1-2C)alkyl group is methyl or ethyl; for a (1-3C)alkyl group is methyl, ethyl, propyl or isopropyl; for a (1-4C)alkyl group is methyl, ethyl, propyl, isopropyl or butyl; for a (1-5C)alkyl group is methyl, ethyl, propyl, isopropyl, butyl or pentyl; and for a (1-4C)alkoxy group is methoxy, ethoxy, isopropoxy or t-butoxy.
A compound of formula I may be made by processes which include processes known in the chemical art for the production of known compounds of formula I or of structurally analogous compounds or by a novel process described herein. A process for a novel compound of formula I (or a pharmaceutically acceptable salt thereof), novel processes for a compound of formula I and novel intermediates for the manufacture of a compound of formula I as defined above provide further features of the invention and are illustrated by the following procedures in which the meanings of the generic radicals are as defined above, unless otherwise specified. It will be recognized that it may be preferred or necessary to prepare a compound of formula I in which a functional group is protected using a conventional protecting group, then to remove the protecting group to provide the compound of formula I.
In general, a compound of formula I may be prepared according to one of the routes outlined in Scheme I, illustrating the preparation of a compound of formula I in which there is a group Q at the 5-position, and described in the examples, in which each of Q, Q2, Q3 and Q5, respectively, represents a value defined for the groups Q, R2, R3 and R5, a protected version of such a group, or moiety which can be further elaborated into such a group. Final conversion of a group Q, Q2, Q3 or Q5 into R, R2, R3 or R5 is carried out at a convenient point, consistent with the chemistry employed. 
Thus, there is provided a process for preparing a novel compound of formula I (or a pharmaceutically acceptable salt thereof) as provided in any of the above descriptions which is selected from any of those described in the examples, including,
cyclizing a corresponding amide of formula II 
xe2x80x83(or for a compound of formula Ia, a compound of formula IIa) 
xe2x80x83by heating it, for example in an inert solvent as described in the Examples below;
whereafter, for any of the above procedures, when a functional group is protected using a protecting group, removing the protecting group;
whereafter, for any of the above procedures, when a pharmaceutically acceptable salt of a compound of formula I is required, it may be obtained by reacting the basic or acidic form of such a compound of formula I with an acid or base affording a physiologically acceptable counterion or by any other conventional procedure.
Novel intermediate or starting material compounds, such as an amide of formula II provide a further aspect of the invention.
As mentioned above, a compound corresponding to a compound of formula I but in which a functional group is protected may serve as an intermediate for a compound of formula I. Accordingly, such protected intermediates for a novel compound of formula I provide further aspects of the invention. Thus, as one particular aspect of the invention, there is provided a compound corresponding to a novel compound of formula I as defined above in which R (for example as R5) is hydroxy, but in which the corresponding substituent is xe2x80x94ORp in place of hydroxy, wherein Rp is a phenol protecting group other than methyl. Phenol protecting groups are well known in the art, for example as described in T. W. Greene and P. G. M. Wuts, xe2x80x9cProtecting Groups in Organic Synthesisxe2x80x9d (1991). Particular values of Rp include, for example, benzyl and allyl. Further, Rp may denote a functionalized resin, for example as disclosed in H. V. Meyers, et al., Molecular Diversity, (1995), 1, 13-20.
As mentioned above, the invention includes pharmaceutically acceptable salts of the thrombin inhibiting compounds defined by the above formula I. A particular compound of this invention which possesses one or more sufficiently basic functional groups will react with any of a number of inorganic and organic acids affording a physiologically acceptable counterion to form a pharmaceutically acceptable salt. Acids commonly employed to form pharmaceutically acceptable acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such pharmaceutically acceptable salts thus are the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycollate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like. Preferred pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid, hydrobromic acid and sulfuric acid.
A compound of formula I which is acidic forms salts with pharmaceutically acceptable bases. Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceutically acceptable cation, which includes alkalai metal salts (especially sodium and potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum salts and ammonium salts, as well as salts made from physiologically acceptable organic bases such as triethylamine, morpholine, piperidine and triethanolamine. The potassium and sodium salt forms are particularly preferred.
If not commercially available, the necessary starting materials for the preparation of a compound of formula I may be prepared by procedures which are selected from standard techniques of organic chemistry, including aromatic and heteroaromatic substitution and transformation, from techniques which are analogous to the syntheses of known, structurally similar compounds, and techniques which are analogous to the above described procedures or procedures described in the Examples. It will be clear to one skilled in the art that a variety of sequences is available for the preparation of the starting materials. Starting materials which are novel provide another aspect of the invention.
Selective methods of protection and deprotection are well known in the art for preparation of compounds such as those of formula I discussed above.
Generally, the compounds of the invention are isolated best in the form of acid addition salts. Salts of the compounds of formula I formed with acids such as those mentioned above are useful as pharmaceutically acceptable salts for administration of the antithrombotic agents and for preparation of formulations of these agents. Other acid addition salts may be prepared and used in the isolation and purification of the compounds.
As noted above, the optically active isomers and diastereomers of the compounds of formula I are also considered part of this invention. Such optically active isomers may be prepared from their respective optically active precursors by the procedures described above, or by resolving the racemic mixtures. This resolution can be carried out by derivatization with a chiral reagent followed by chromatography or by repeated crystallization. Removal of the chiral auxiliary by standard methods affords substantially optically pure isomers of the compounds of the present invention or their precursors. Further details regarding resolutions can be obtained in Jacques, et al., Enantiomers, Racemates, and Resolutions, John Wiley and Sons, 1981.
The compounds of the invention are believed to selectively inhibit thrombin over other proceinases and nonenzyme proteins involved in blood coagulation without appreciable interference with the body""s natural clot lysing ability (the compounds have a low inhibitory effect on fibrinolysis). Further, such selectivity is believed to permit use with thrombolytic agents without substantial interference with thrombolysis and fibrinolysis.
The invention in one of its aspects provides a method of inhibiting thrombin in mammals comprising administering to a mammal in need of treatment an effective (thrombin inhibiting) dose of a compound of formula I.
In another of its aspects, the invention provides a method of treating a thromboembolic disorder comprising administering to a mammal in need of treatment an effective (thromboembolic disorder therapeutic and/or prophylactic amount) dose of a compound of formula I.
The invention in another of its aspects provides a method of inhibiting coagulation in mammals comprising administering to a mammal in need of treatment an effective (coagulation inhibiting) dose of a compound of formula I.
The thrombin inhibition, coagulation inhibition and thromboembolic disorder treatment contemplated by the present method includes both medical therapeutic and/or prophylactic treatment as appropriate.
In a further embodiment the invention relates to treatment, in a human or animal, of conditions where inhibition of thrombin is required. The compounds of the invention are expected to be useful in animals, including man, in treatment or prophylaxis of thrombosis and hypercoagulability in blood and tissues. Disorders in which the compounds have a potential utility are in treatment or prophylaxis of thrombosis and hypercoagulability in blood and tissues. Disorders in which the compounds have a potential utility, in treatment and/or prophylaxis, include venous thrombosis and pulmonary embolism, arterial thrombosis, such as in myocardial ischemia, myocardial infarction, unstable angina, thrombosis-based stroke and peripheral arterial thrombosis. Further, the compounds have expected utility in the treatment or prophylaxis of atherosclerotic disorders (diseases) such as coronary arterial disease, cerebral arterial disease and peripheral arterial disease. Further, the compounds are expected to be useful together with thrombolytics in myocardial infarction. Further, the compounds have expected utility in prophylaxis for reocclusion after thrombolysis, percutaneous transluminal angioplasty (PTCA) and coronary bypass operations. Further, the compounds have expected utility in prevention of rethrombosis after microsurgery. Further, the compounds are expected to be useful in anticoagulant treatment in connection with artificial organs and cardiac valves. Further, the compounds have expected utility in anticoagulant treatment in hemodialysis and disseminated intravascular coagulation. A further expected utility is in rinsing of catheters and mechanical devices used in patients in vivo, and as an anticoagulant for preservation of blood, plasma and other blood products in vitro. Still further, the compounds have expected utility in other diseases where blood coagulation could be a fundamental contributing process or a source of secondary pathology, such as cancer, including metastasis, inflammatory diseases, including arthritis, and diabetes. The anti-coagulant compound is administered orally, parenterally e.g. by intravenous infusion (iv), intramuscular injection (im) or subcutaneously (sc).
The specific dose of a compound administered according to this invention to obtain therapeutic and/or prophylactic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the compound administered, the rate of administration, the route of administration, and the condition being treated.
A typical daily dose for each of the above utilities is between about 0.01 mg/kg and about 1000 mg/kg. The dose regimen may vary e.g. for prophylactic use a single daily dose may be administered or multiple doses such as 3 or 5 times daily may be appropriate. In critical care situations a compound of the invention is administered by iv infusion at a rate between about 0.01 mg/kg/h and about 20 mg/kg/h and preferably between about 0.1 mg/kg/h and about 5 mg/kg/h.
The method of this invention also is practiced in conjunction with a clot lysing agent e.g. tissue plasminogen activator (t-PA), modified t-PA, streptokinase or urokinase. In cases when clot formation has occurred and an artery or vein is blocked, either partially or totally, a clot lysing agent is usually employed. A compound of the invention can be administered prior to or along with the lysing agent or subsequent to its use, and preferably further is administered along with aspirin to prevent the reoccurrence of clot formation.
The method of this invention is also practiced in conjunction with a platelet glycoprotein receptor (IIb/IIIa) antagonist, that inhibits platelet aggregation. A compound of the invention can be administered prior to or along with the IIb/IIIa antagonist or subsequent to its use to prevent the occurrence or reoccurrence of clot formation.
The method of this invention is also practiced in conjunction with aspirin. A compound of the invention can be administered prior to or along with aspirin or subsequent to its use to prevent the occurrence or reoccurrence of clot formation. As stated above, preferably a compound of the present invention is administered in conjunction with a clot lysing agent and aspirin.
This invention also provides pharmaceutical formulations for use in the above described therapeutic method. Pharmaceutical formulations of the invention comprise an effective thrombin inhibiting amount of a compound of formula I in association with a pharmaceutically acceptable carrier, excipient or diluent. For oral administration the antithrombotic compound is formulated in gelatin capsules or tablets which may contain excipients such as binders, lubricants, disintegration agents and the like. For parenteral administration the antithrombotic is formulated in a pharmaceutically acceptable diluent e.g. physiological saline (0.9 percent), 5 percent dextrose, Ringer""s solution and the like.
The compound of the present invention can be formulated in unit dosage formulations comprising a dose between about 0.1 mg and about 1000 mg. Preferably the compound is in the form of a pharmaceutically acceptable salt such as for example the sulfate salt, acetate salt or a phosphate salt. An example of a unit dosage formulation comprises 5 mg of a compound of the present invention as a pharmaceutically acceptable salt in a 10 mL sterile glass ampoule. Another example of a unit dosage formulation comprises about 10 mg of a compound of the present invention as a pharmaceutically acceptable salt in 20 mL of isotonic saline contained in a sterile ampoule.
The compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. The compounds of the present invention are preferably formulated prior to administration. Another embodiment of the present invention is a pharmaceutical formulation comprising an effective amount of a novel compound of formula I or a pharmaceutically acceptable salt or solvate thereof in association with a pharmaceutically acceptable carrier, diluent or excipient therefor.
The active ingredient in such formulations comprises from 0.1 percent to 9.9 percent by weight of the formulation. By xe2x80x9cpharmaceutically acceptablexe2x80x9d it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
The present pharmaceutical formulations are prepared by known procedures using well known and readily available ingredients. The compositions of this invention may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art. In making the compositions of the present invention, the active ingredient will usually be admixed with a carrier, or diluted by a carrier, or enclosed within a carrier which may be in the form of a capsule, sachet, paper or other container. When the carrier serves as a diluent, it may be a solid, semi-solid or liquid material which acts as a vehicle, excipient or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, (as a solid or in a liquid medium), soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile packaged powders, and the like.
The following formulation examples are illustrative only and are not intended to limit the scope of the invention in any way. xe2x80x9cActive ingredient,xe2x80x9d of course, means a compound according to formula I or a pharmaceutically acceptable salt or solvate thereof.
The active compound is mixed with ethanol and the mixture added to a portion of the propellant 22, cooled to xe2x88x9230xc2x0 C. and transferred to a filling device. The required amount is then fed to a stainless steel container and diluted with the remainder of the propellant. The valve units are then fitted to the container.
The active ingredient, starch and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly. The aqueous solution containing polyvinylpyrrolidone is mixed with the resultant powder, and the mixture then is passed through a No. 14 mesh U.S. sieve. The granules so produced are dried at 50xc2x0 C. and passed through a No. 18 mesh U.S. Sieve. The sodium carboxymethyl starch, magnesium stearate and talc, previously passed through a No. 60 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 150 mg.
The active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No. 45 mesh U.S. sieve, and filled into hard gelatin capsules in 200 mg quantities.
The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2 g capacity and allowed to cool.
The active ingredient is passed through a No. 45 mesh U.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form a smooth paste. The benzoic acid solution, flavor and color are diluted with a portion of the water and added, with stirring. Sufficient water is then added to produce the required volume.
The solution of the above ingredients generally is administered intravenously to a subject at a rate of 1 mL per minute.
The ability of the compounds of the present invention to be an effective and orally active thrombin inhibitor are evaluated in one or more of the following assays.
The compounds provided by the invention (formula I) selectively inhibit the action of thrombin in mammals. The inhibition of thrombin is demonstrated by in vitro inhibition of the amidase activity of thrombin as measured in an assay in which thrombin hydrolyzes the chromogenic substrate, N-benzoyl-L-phenylalanyl-L-valyl-L-arginyl-p-nitroanilide, N-benzoyl-L-Phe-L-Val-L-Arg-p-nitroanilide.
The assay is carried out by mixing 50 xcexcL buffer (0.03M Tris, 0.15M NaCl, pH 7.4) with 25 xcexcL of human thrombin solution (purified human thrombin, Enzyme Research Laboratories, South Bend, Ind., at 8 NIH units/mL) and 25 xcexcL of test compound in a solvent (50% aqueous methanol (v:v)). Then 150 xcexcL of an aqueous solution of the chromogenic substate (at 0.25 mg/mL) are added and the rates of hydrolysis of the substrate are measured by monitoring the reactions at 405 nm for the release of p-nitroaniline. Standard curves are constructed by plotting free thrombin concentration against hydrolysis rate. The hydrolysis rates observed with test compounds are then converted to xe2x80x9cfree thrombinxe2x80x9d values in the respective assays by use of the standard curves. The bound thrombin (bound to test compound) is calculated by subtracting the amount of free thrombin observed in each assay from the known initial amount of thrombin used in the assay. The amount of free inhibitor in each assay is calculated by subtracting the number of moles of bound thrombin from the number of moles of added inhibitor (test compound).
The Kass value is the hypothetical equilibrium constant for the reaction between thrombin and the test compound (I).             Thrombin      +      I        ⇄          Thrombin      -      I            Kass    =                  [                  Thrombin          -          I                ]                    [                              (            Thrombin            )                    xc3x97                      (            I            )                          ]            
Kass is calculated for a range of concentrations of test compounds and the mean value reported in units of liter per mole. In general, a thrombin inhibiting compound of formula I of the instant invention exhibits a Kass of 0.05xc3x97106 L/mole or much greater.
By substantially following the procedures described above for human thrombin, and using other human blood coagulation system serine proteases and using fibrinolytic system serine proteases, with the appropriate chromogenic substrates, identified below, the selectivity of the compounds of the present invention with respect to the coagulation factor serine proteases and to the fibronolytic serine proteases are evaluated as well as their substantial lack of interference with human plasma clot fibrinolysis.
Human factors X, Xa, IXa, XIa, and XIIa are purchased from Enzyme Research Laboratories, South Bend, Ind.; human urokinase from Leo Pharmaceuticals, Denmark; and recombinant activated Protein C (aPC) is prepared at Eli Lilly and Co. substantially according to U.S. Pat. No. 4,981,952. Chromogenic substrates: N-Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (for factor Xa); N-Cbz-D-Arg-Gly-Arg-p-nitroanilide (for factor IXa assay as the factor Xa substrate); Pyroglutamyl-Pro-Arg-p-nitroanilide (for Factor XIa and for aPC); H-D-Pro-Phe-Arg-p-nitroanilide (for factor XIIa); and Pyroglutamyl-Gly-Arg-p-nitroanilide (for urokinase); are purchased from Kabi Vitrum, Stockholm, Sweden, or from Midwest Biotech, Fishers, Ind. Bovine trypsin is purchased from Worthington Biochemicals, Freehold, N.J., and human plasma kallikrein from Kabi Vitrum, Stockholm, Sweden. Chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide for plasma kallikrein is purchased from Kabi Vitrum, Stockholm, Sweden. N-Benzoyl-Phe-Val-Arg-p-nitroanilide, the substrate for human thrombin and for trypsin, is synthesized according to procedures described above for the compounds of the present invention, using known methods of peptide coupling from commercially available reactants, or purchased from Midwest Biotech, Fishers, Ind.
Human plasmin is purchased from Boehringer Mannheim, Indianapolis, Ind.; nt-PA is purchased as single chain activity reference from American Diagnostica, Greenwich, Conn.; modified-t-PA6 (mt-PA6) is prepared at Eli Lilly and Company by procedure known in the art (See, Burck, et al., J. Biol. Chem., 265, 5120-5177 (1990). Plasmin chromogenic substrate H-D-Val-Leu-Lys-p-nitroanilide and tissue plasminogen activator (t-PA) substrate H-D-Ile-Pro-Arg-p-nitroanilide are purchased from Kabi Vitrum, Stockholm, Sweden.
In the chromogenic substrates described above the three-letter symbols Ile, Glu, Gly, Pro, Arg, Phe, Val, Leu and Lys are used to indicate the corresponding amino acid group isoleucine, glutamic acid, glycine, proline, arginine, phenylalanine, valine, leucine and lysine, respectively.
Thrombin inhibitors preferably should spare fibrinolysis induced by urokinase, tissue plasminogen activator (t-PA) and steptokinase. This would be important to the therapeutic use of such agents as an adjunct to streptokinase, t-PA or urokinase thrombolytic therapy and to the use of such agents as an endogenous fibrinolysis-sparing (with respect to t-PA and urokinase) antithrombotic agents. In addition to the lack of interference with the amidase activity of the fibrinolytic proteases, such fibrinolytic system sparing can be studied by the use of human plasma clots and their lysis by the respective fibrinolytic plasminogen activators.
Materials
Dog plasma is obtained from conscious mixed-breed hounds (either sex Butler Farms, Clyde, N.Y., U.S.A.) by venipuncture into 3.8 percent citrate. Fibrinogen is prepared from fresh dog plasma and human fibrinogen is prepared from in-date ACD human blood at the fraction I-2 according to previous procedures and specifications. Smith, Biochem. J., 185, 1-11 (1980); and Smith, et al., Biochemistry, 11, 2958-2967, (1972). Human fibrinogen (98 percent pure/plasmin free) is from American Diagnostica, Greenwich, Conn. Radiolabeling of fibrinogen I-2 preparations is performed as previously reported. Smith, et al., Biochemistry, 11, 2958-2967, (1972). Urokinase is purchased from Leo Pharmaceuticals, Denmark, as 2200 Ploug units/vial. Streptokinase is purchased from Hoechst-Roussel Pharmaceuticals, Somerville, N.J.
Methodsxe2x80x94Effects on Lysis of Human Plasma Clots by t-PA
Human plasma clots are formed in micro test tubes by adding 50 xcexcL thrombin (73 NIH unit/mL) to 100 xcexcL human plasma which contains 0.0229 xcexcCi 125-iodine labeled fibrinogen. Clot lysis is studied by overlaying the clots with 50 xcexcL of urokinase or streptokinase (50, 100, or 1000 unit/mL) and incubating for 20 hours at room temperature. After incubation the tubes are centrifuged in a Beckman Microfuge. 25 xcexcL of supernate is added into 1.0 mL volume of 0.03 M tris/0.15 M NaCl buffer for gamma counting. Counting controls 100 percent lysis are obtained by omitting thrombin (and substituting buffer). The thrombin inhibitors are evaluated for possible interference with fibrinolysis by including the compounds in the overlay solutions at 1, 5, and 10 xcexcg/mL concentrations. Rough approximations of IC50 values are estimated by linear extrapolations from data points to a value which would represent 50 percent of lysis for that particular concentration of fibrinolytic agent.
Anticoagulant Activity
Materials
Dog plasma and rat plasma are obtained from conscious mixed-breed hounds (either sex, Butler Farms, Clyde, N.Y., U.S.A.) or from anesthetized male Sprague-Dawley rats (Harlan Sprague-Dawley, Inc., Indianapolis, Ind., U.S.A.) by venipuncture into 3.8 percent citrate. Fibrinogen is prepared from in-date ACD human blood as the fraction I-2 according to previous procedures and specifications. Smith, Biochem. J., 185, 1-11 (1980); and Smith, et al., Biochemistry, 11, 2958-2967 (1972). Human fibrinogen is also purchased as 98 percent pure/plasmin free from American Diagnostica, Greenwich, Conn. Coagulation reagents Actin, Thromboplastin, Innovin and Human plasma are from Baxter Healthcare Corp., Dade Division, Miami, Fla. Bovine thrombin from Parke-Davis (Detroit, Mich.) is used for coagulation assays in plasma.
Methods
Anticoagulation Determinations
Coagulation assay procedures are as previously described. Smith, et al., Thrombosis Research, 50, 163-174 (1988). A CoAScreener coagulation instrument (American LABor, Inc.) is used for all coagulation assay measurements. The prothrombin time (PT) is measured by adding 0.05 mL saline and 0.05 mL Thromboplastin-C reagent or recombinant human tissue factor reagent (Innovin) to 0.05 mL test plasma. The activated partial thromboplastin time (APTT) is measured by incubation of 0.05 mL test plasma with 0.05 mL Actin reagent for 120 seconds followed by 0.05 mL CaCl2 (0.02 M). The thrombin time (TT) is measured by adding 0.05 mL saline and 0.05 mL thrombin (10 NIH units/mL) to 0.05 mL test plasma. The compounds of formula I are added to human or animal plasma over a wide range of concentrations to determine prolongation effects on the APTT, PT, and TT assays. Linear extrapolations are performed to estimate the concentrations required to double the clotting time for each assay.
Animals
Male Sprague Dawley rats (350-425 gm, Harlan Sprague Dawley Inc., Indianapolis, Ind.) are anesthetized with xylazine (20 mg/kg, s.c.) and ketamine (120 mg/kg, s.c.) and maintained on a heated water blanket (37xc2x0 C.). The jugular vein(s) is cannulated to allow for infusions.
Arterio-Venous Shunt Model
The left jugular vein and right carotid artery are cannulated with 20 cm lengths of polyethylene PE 60 tubing. A 6 cm center section of larger tubing (PE 190) with a cotton thread (5 cm) in the lumen, is friction fitted between the longer sections to complete the arterio-venous shunt circuit. Blood is circulated through the shunt for 15 min before the thread is carefully removed and weighed. The weight of a wet thread is subtracted from the total weight of the thread and thrombus (see J. R. Smith, Br J Pharmacol, 77:29, 1982). In this model preferred compounds of the instant invention reduce the net clot weight to approximately 25-30% of control, or even lower, at an i.v. dose of 33.176 xcexcmol/kg/h.
FeCl3 Model of Arterial Injury
The carotid arteries are isolated via a midline ventral cervical incision. A thermocouple is placed under each artery and vessel temperature is recorded continuously on a strip chart recorder. A cuff of tubing (0.058 IDxc3x970.077 ODxc3x974 mm, Baxter Med. Grade Silicone), cut longitudinally, is placed around each carotid directly above the thermocouple. FeCl3 hexahydrate is dissolved in water and the concentration (20 percent) is expressed in terms of the actual weight of FeCl3 only. To injure the artery and induce thrombosis, 2.85 xcexcL is pipetted into the cuff to bathe the artery above the thermocouple probe. Arterial occlusion is indicated by a rapid drop in temperature. The time to occlusion is reported in minutes and represents the elapsed time between application of FeCl3 and the rapid drop in vessel temperature (see K. D. Kurz, Thromb. Res., 60:269, 1990).
Spontaneous Thrombolysis Model
In vitro data suggests that thrombin inhibitors inhibit thrombin and, at higher concentrations, may inhibit other serine proteases, such as plasmin and tissue plasminogen activator. To assess if the compounds inhibit fibrinolysis in vivo, the rate of spontaneous thrombolysis is determined by implanting a labeled whole blood clot into the pulmonary circulation. Rat blood (1 mL) is mixed rapidly with bovine thrombin (4 IU, Parke Davis) and 125I human Fibrogen (5 xcexcCi, ICN), immediately drawn into silastic tubing and incubated at 37xc2x0 C. for 1 hour. The aged thrombus is expelled from the tubing, cut into 1 cm segments, washed 3xc3x97 in normal saline and each segment is counted in a gamma counter. A segment with known counts is aspirated into a catheter that is subsequently implanted into the jugular vein. The catheter tip is advanced to the vicinity of the right atrium and the clot is expelled to float into the pulmonary circulation. One hour after implant, the heart and lungs are harvested and counted separately. Thrombolysis is expressed as a percentage where:       %    ⁢          xe2x80x83        ⁢    Thrombolysis    =                    (                              injected            ⁢                          xe2x80x83                        ⁢            cpm                    -                      lung            ⁢                          xe2x80x83                        ⁢            cpm                          )                    injected        ⁢                  xe2x80x83                ⁢        cpm              xc3x97    100  
The fibrinolytic dissolution of the implanted clot occurs time-dependently (see J. P. Clozel, Cardiovas. Pharmacol., 12:520, 1988).
Coagulation Parameters
Plasma thrombin time (TT) and activated partial thromboplastin time (APTT) are measured with a fibrometer. Blood is sampled from a jugular catheter and collected in syringe containing sodium citrate (3.8 percent, 1 part to 9 parts blood). To measure TT, rat plasma (0.1 mL) is mixed with saline (0.1 mL) and bovine thrombin (0.1 mL, 30 U/mL in TRIS buffer; Parke Davis) at 37xc2x0 C. For APTT, plasma (0.1 mL) and APTT solution (0.1 mL, Organon Teknika) are incubated for 5 minutes (37xc2x0 C.) and CaCl2 (0.1 mL, 0.025 M) is added to start coagulation. Assays are done in duplicate and averaged.
Index of Bioayailability
For a measure of bioactivity, plasma thrombin time (TT) serves as a substitute for the assay of parent compound on the assumption that observed increments in TT resulted from thrombin inhibition by parent only. The time course of the effect of the thrombin inhibitor upon TT is determined after i.v bolus administration to anesthetized rats and after oral treatment of fasted conscious rats. Due to limitations of blood volume and the number of points required to determine the time course from time of treatment to the time when the response returns to pretreatment values, two populations of rats are used. Each sample population represents alternating sequential time points. The average TT over the time course is used to calculate area under the curve (AUC). The index of bioavailability is calculated by the formula shown below and is expressed as percent relative activity.
The area under the curve (AUC) of the plasma TT time course is determined and adjusted for the dose. This index of bioavailability is termed xe2x80x9c% Relative Activityxe2x80x9d and is calculated as       %    ⁢          xe2x80x83        ⁢    Relative    ⁢          xe2x80x83        ⁢    Activity    =                    AUC        ⁢                  xe2x80x83                ⁢        po                    AUC        ⁢                  xe2x80x83                ⁢        iv              xc3x97          xe2x80x83        ⁢                  Dose        ⁢                  xe2x80x83                ⁢        iv                    Dose        ⁢                  xe2x80x83                ⁢        po              xc3x97    100  
Compounds
Compound solutions are prepared fresh daily in normal saline and are injected as a bolus or are infused starting 15 minutes before and continuing throughout the experimental perturbation which is 15 minutes in the arteriovenous shunt model and 60 minutes in the FeCl3 model of arterial injury and in the spontaneous thrombolysis model. Bolus injection volume is 1 mL/kg for i.v., and 5 mL/kg for p.o., and infusion volume is 3 mL/hr.
Statistics
Results are expressed as means +/xe2x88x92SEM. One-way analysis of variance is used to detect statistically significant differences and then Dunnett""s test is applied to determine which means are different. Significance level for rejection of the null hypothesis of equal means is P less than 0.05.
Animals
Male dogs (Beagles; 18 months-2 years; 12-13 kg, Marshall Farms, North Rose, N.Y. 14516) are fasted overnight and fed Purina certified Prescription Diet (Purina Mills, St. Louis, Mo.) 240 minutes after dosing. Water is available ad libitum. The room temperature is maintained between 66-74xc2x0 F.; 45-50 percent relative humidity; and lighted from 0600-1800 hours.
Pharmacokinetic Model
Test compound is formulated immediately prior to dosing by dissolving in sterile 0.9 percent saline to a 5 mg/mL preparation. Dogs are given a single 2 mg/kg dose of test compound by oral gavage. Blood samples (4.5 mL) are taken from the cephalic vein at 0.25, 0.5, 0.75, 1, 2, 3, 4 and 6 hours after dosing. Samples are collected in citrated Vacutainer tubes and kept on ice prior to reduction to plasma by centrifugation. Plasma samples are analyzed by HPLC MS. Plasma concentration of test compound is recorded and used to calculate the pharmacokinetic parameters: elimination rate constant, Ke; total clearance, Clt; volume of distribution, VD; time of maximum plasma test compound concentration, Tmax; maximum concentration of test compound of Tmax, Cmax; plasma half-life, t0.5; and area under the curve, A.U.C.; fraction of test compound absorbed, F.
Canine Model of Coronary Artery Thrombosis
Surgical preparation and instrumentation of the dogs are as described in Jackson, et al., Circulation, 82, 930-940 (1990). Mixed-breed hounds (aged 6-7 months, either sex, Butler Farms, Clyde, N.Y., U.S.A.) are anesthetized with sodium pentobarbital (30 mg/kg intravenously, i.v.), intubated, and ventilated with room air. Tidal volume and respiratory rates are adjusted to maintain blood PO2, PCO2, and pH within normal limits. Subdermal needle electrodes are inserted for the recording of a lead II ECG.
The left jugular vein and common carotid artery are isolated through a left mediolateral neck incision. Arterial blood pressure (ABP) is measured continuously with a precalibrated Millar transducer (model (MPC-500, Millar Instruments, Houston, Tex., U.S.A.) inserted into the carotid artery. The jugular vein is cannulated for blood sampling during the experiment. In addition, the femoral veins of both hindlegs are cannulated for administration of test compound.
A left thoracotomy is performed at the fifth intercostal space, and the heart is suspended in a pericardial cradle. A 1- to 2-cm segment of the left circumflex coronary artery (LCX) is isolated proximal to the first major diagonal ventricular branch. A 26-gauge needle-tipped wire anodal electrode (Teflon-coated, 30-gauge silverplated copper wire) 3-4 mm long is inserted into the LCX and placed in contact with the intimal surface of the artery (confirmed at the end of the experiment). The stimulating circuit is completed by placing the cathode in a subcutaneous (s.c.) site. An adjustable plastic occluder is placed around the LCX, over the region of the electrode. A precalibrated electromagnetic flow probe (Carolina Medical Electronics, King, N.C., U.S.A.) is placed around the LCX proximal to the anode for measurement of coronary blood flow (CBF). The occluder is adjusted to produce a 40-50 percent inhibition of the hyperemic blood flow response observed after 10-s mechanical occlusion of the LCX. All hemodynamic and ECG measurements are recorded and analyzed with a data acquisition system (model M3000, Modular Instruments, Malvern, Pa. U.S.A.).
Thrombus Formation and Compound Administration Regimens
Electrolytic injury of the intima of the LCX is produced by applying 100-xcexcA direct current (DC) to the anode. The current is maintained for 60 min and then discontinued whether the vessel has occluded or not. Thrombus formation proceeds spontaneously until the LCX is totally occluded (determined as zero CBF and an increase in the S-T segment). Compound administration is started after the occluding thrombus is allowed to age for 1 hour. A 2-hour infusion of the compounds of the present invention at doses of 0.5 and 1 mg/kg/hour is begun simultaneously with an infusion of thrombolytic agent (e.g. tissue plasminogen activator, streptokinase, APSAC). Reperfusion is followed for 3 hour after administration of test compound. Reocclusion of coronary arteries after successful thrombolysis is defined as zero CBF which persisted for at least 30 minutes.
Hematolocry and Template Bleeding Time Determinations
Whole blood cell counts, hemoglobin, and hematocrit values are determined on a 40-xcexcL sample of citrated (3.8 percent) blood (1 part citrate:9 parts blood) with a hematology analyzer (Cell-Dyn 900, Sequoia-Turner. Mount View, Calif., U.S.A.). Gingival template bleeding times are determined with a Simplate II bleeding time device (Organon Teknika Durham, N.C., U.S.A.). The device is used to make 2 horizontal incisions in the gingiva of either the upper or lower left jaw of the dog. Each incision is 3 mm widexc3x972 mm deep. The incisions are made, and a stopwatch is used to determine how long bleeding occurs. A cotton swab is used to soak up the blood as it oozes from the incision. Template bleeding time is the time from incision to stoppage of bleeding. Bleeding times are taken just before administration of test compound (0 min), 60 min into infusion, at conclusion of administration of the test compound (120 min), and at the end of the experiment.
All data are analyzed by one-way analysis of variance (ANOVA) followed by Student-Neuman-Kuels post hoc t test to determine the level of significance. Repeated-measures ANOVA are used to determine significant differences between time points during the experiments. Values are determined to be statistically different at least at the level of p less than 0.05. All values are meanxc2x1SEM. All studies are conducted in accordance with the guiding principles of the American Physiological Society. Further details regarding the procedures are described in Jackson, et al., J. Cardiovasc. Pharmacol., (1993), 21, 587-599.
The following Examples are provided to further describe the invention and are not to be construed as limitations thereof.
The abbreviations, symbols and terms used in the examples have the following meanings.
Ac=acetyl
AIBN=azobisisobutyronitrile
Anal.=elemental analysis
Bn or Bzl=benzyl
Bu=butyl
n-BuLi=butyllithium
calcd=calculated
DCC=dicyclohexylcarbodiimide
DIBAL-H=diisobutyl aluminum hydride
DMF=dimethylformamide
DMSO=dimethylsulfoxide
Et=ethyl
EtOAc=ethyl acetate
Et3N=triethylamine
Et2O=diethyl ether
EtOH=ethanol
EtSH=ethanethiol
FAB=Fast Atom Bombardment (Mass Spectrascopy)
FDMS=field desorption mass spectrum
Hex=hexanes
HOAt=1-hydroxy-7-azabenzotriazole
HPLC=High Performance Liquid Chromatography
HRMS=high resolution mass spectrum
i-PrOH=isopropanol
IR=Infrared Spectrum
LAH=lithium aluminum hydride
Me=methyl
MeI=methyl iodide
MeOH=methanol
MPLC=Medium Pressure Liquid Chromatography
NBS=N-bromosuccinimide
NMR=Nuclear Magnetic Resonance
Ph=phenyl
PPA=polyphosphoric acid
i-Pr=isopropyl
Rochelle""s Salt=potassium sodium tartrate
RPHPLC=Reversed Phase High Performance Liquid Chromatography
SiO2=silica gel
SM=starting material
TBS=tert-butyldimethylsilyl
TEA=triethylamine
Temp.=temperature
TFA=trifluoroacetic acid
THF=tetrahydrofuran
TIPS=triisopropylsilyl
TLC=thin layer chromatography
triflic acid=trifluoromethanesulfonic acid
Unless otherwise stated, pH adjustments and work up are with aqueous acid or base solutions. PrepLC indicates preparative liquid chromatography using xe2x80x9cPrep Pak (TM)xe2x80x9d silica cartridges; radial chromatography indicates preparative chromatography using a xe2x80x9cChromatotron (TM)xe2x80x9d instrument.